Human immunodeficiency virus type 1 (HIV-1) virions are released from cells as immature, noninfectious particles that must subsequently undergo proteolytic maturation to become infectious. Entry of virions into new target cells occurs through a membrane fusion reaction mediated by the Env glycoproteins gp120 and gp41. Although the mechanism of Env-catalyzed membrane fusion is relatively well characterized, less clear is the role of the 152 amino acid gp41 cytoplasmic domain in Env-mediated fusion. We have recently discovered that immature HIV-1 particles are inhibited for fusion through a mechanism involving the gp41 cytoplasmic tail (CT). Truncation of the tail or cleavage of Pr55Gag between the MA and NC domains, activates HIV-1 particles for fusion. This novel mechanism for regulating fusion may promote HIV-1 replication by enhancing productive entry of the virus, in addition, by inhibiting Env conformationat changes until the core has matured, the gp41-Gag interaction may confer a replicative advantage by limiting exposure of Env epitopes recognized by neutralizing antibodies. We propose here a series of six highly focused Specific Aims to dissect the mechanism of immature HIV-1 fusion inhibition and to determine the consequences to HIV-1 replication. Specifically, we will: 1. Determine whether maturation-dependent HIV-1 fusion extends to R5-tropic Env and Gag proteins from HIV-1 primary isolates; 2. Map the regions of the gp41 CT required for fusion inhibition; 3. Determine the role of lipid rafts in coupling HIV-1 fusion to virion maturation; 4. Quantify receptor-induced conformationat changes on immature HIV-1 particles; 5. Determine the role of viral RNA in fusion inhibition; and 6. Evaluate the role of the gp41 CT in conferring resistance to neutralizing antibodies during continuous HIV-1 replication. These studies will reveal new fundamental insights into the regulation of HIV-t entry and the role of the gp41 cytoplasmic tail in HIV-1 replication.